Glucose transport from blood to brain intracellular spaces is limited by transport through the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo. Recent studies have shown that more than 90% of BBB glucose transport is mediated by the GLUT1 isoform of the sodium independent glucose transporter gene family. The proposed studies will examine role of post-transcriptional mechanisms in regulating BBB GLUT1 mRNA. The role of cytosolic factors that alter GLUT1 mRNA stability in hypoglycemia and in development will be examined. In addition, full-length GLUT1 mRNA will be used in cell-free translation assays to investigate the effects of brain capillary-derived cytosolic proteins in regulating GLUT1 translation efficiency, which prior work has indicated may be impaired at certain periods of development, and in high- grade human glioma cytosolic proteins to identify a novel cis/trans interaction between the GLUT1 3-UTR and a 77 kDa cytosolic protein. The sequence of the GLUT1 3-UTR containing the cis element was localized with RNase T1 mapping experiments, and oligodeoxy-nucleotide competitor studies. The molecular weight of the cytosolic protein interacting with the cis element of the 3'-UTR was identified by ultraviolet (UV) light cross-linking of RNA/cytosol protein complexes. The region spanning the cis element is conserved in GLUT1 of all species, but is not found in other mRNA sequences in GenBank. The identification of this cis/trans- mechanism allows for an examination of its functional role related to either mRNA stability or mRNA translation efficiency. GLUT1 gene expression in brain is confined to the brain capillary endothelium. Therefore, GLUT1, in addition to limiting BBB glucose transport, is also a genetic marker of the differentiation state of the BBB, and the induction of barrier properties within the capillary endothelium by brain-derived factors. Measurement of the induction of GLUT1 mRNA or protein in small amounts of brain capillary endothelial cells grown in primary culture under the influence of brain-derived factors. Measurement of the induction of GLUT1 mRNA or protein in small amounts of brain capillary endothelial cells grown in primary culture under the influence of brain-derived factors is now possible with the recent development of quantitative polymerase chain reaction (QPCR) and ELISA assays. Using a cluster dish format and these assays, it is feasible to fractionate and characterize brain fractions that induce GLUT1 levels. Although transplant models have demonstrated the inductive effects of brain-derived proteins on brain capillary endothelium, to date no brain- derived factors that induce BBB specific genes have been identified.